The Hairless Gene
The hairless mouse, a frequent subject of different dermatological experiments, is characterized by hair loss that starts at the age of 14 days postpartum from the upper eyelids and progresses caudally. The process is completed by the age of 3 weeks, when the mice are entirely naked and never grow hair again (Panteleyev et al. 1998b). The phenotype results from proviral integration and consequential aberrant splicing in the mouse hairless gene. Lack of expression of the mouse hairless gene due to inherited mutations leads to the complete loss of hair, known as atrichia. Other mutations have been identified in other mouse hairless alleles, and its human equivalent, resulting in essentially similar phenotypes (Ahmad et al. 1998a; Ahmad et al. 1998b; Panteleyev et al. 1998a). Those studies demonstrated that hairless expression in the hair follicle is necessary for hair cycling, specifically in the transition to the catagen phase.
Catalytic Nucleic Acid Molecules
Gene therapy is perhaps the most exciting promise of modern medical science. The technology of replacing mutant genes with correct ones can provide definitive therapy for a number of diseases. There are, however, conditions—inherited and acquired alike—which cannot be treated by the introduction of a new gene. In many cases, the ablation of an already existing gene may be desirable. In many dominantly inherited diseases, the successful “knock-out” of the mutant gene is, in theory, sufficient to cure the disease. In some other cases, the elimination of a normally functioning, wild-type gene may be necessary for therapeutic gene targeting.
Such is the case in abundant hair growth or hirsutism, in which inhibiting genes which promote hair growth could lead to decreased hair growth and, therefore, improvement. One way to achieve targeted, transient gene suppression is likely going to be through the use of catalytic nucleic acid technology, which includes both ribozymes and DNAzymes.
Ribozymes are RNA structures having a self-catalytic enzymatic function which, together with their sequence-specific and RNA-binding ability, make them capable of cleaving other RNA molecules at specific target sequences (Cech 1987). Recent success has been achieved in engineering ribozymes capable of selectively recognizing target sequences carrying different types of mutations, including single base-pair missense mutations (Parthasarathy et al. 1999; Sioud and Drlica 1991; Vaish et al. 1998).
These encouraging achievements give new perspective to experimental strategies using selective mRNA ablation (Phylactou et al. 1998). The different groups of ribozymes described thus far (including hairpin ribozymes, hammerhead ribozymes and group I intron ribozymes (Bartel 1999)) have different characteristics with respect to their mechanism of splicing, splicing efficiency and target specificity. Several studies have used hammerhead ribozymes to selectively cleave RNA because of the superior target specificity of these ribozymes (Long and Sullenger 1999; Phylactou et al. 1998; Vaish et al. 1998).
Ribozymes can be delivered exogenously, such that the ribozymes are synthesized in vitro. They are usually administered using carrier molecules (Sioud 1996) or without carriers, using ribozymes specially modified to be nuclease-resistant (Flory et al. 1996). The other method is endogenous delivery, in which the ribozymes are inserted into a vector (usually a retroviral vector) which is then used to transfect target cells. There are several possible cassette constructs to chose from (Vaish et al. 1998), including the widely used U1 snRNA expression cassette, which proved to be efficient in nuclear expression of hammerhead ribozymes in various experiments (Bertrand et al. 1997; Michienzi et al. 1996; Montgomery and Dietz 1997).
Recent efforts have led to the successful development of small DNA oligonucleotides that have a structure similar to the hammerhead ribozyme (Santoro and Joyce 1997). These molecules are known as “deoxy-ribozymes”, “deoxyribozymes” and “DNAzymes”, and are virtually DNA equivalents of the hammerhead ribozymes. They consist of a 15 bp catalytic core and two sequence-specific arms with a typical length of 5-13 bp each (Santoro and Joyce 1998). Deoxy-ribozymes have more lenient consensus cleavage site requirements than hammerhead ribozymes, and are less likely to degrade when used for in vivo applications. The most widely used type of these novel catalytic molecules is known as the “10-23” deoxy-ribozyme, whose designation originates from the numbering used by its developers (Santoro and Joyce 1997). Because of their considerable advantages, deoxy-ribozymes have already been used in a wide spectrum of in vitro and in vivo applications (Cairns et al. 2000; Santiago et al. 1999).